We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering Galphai nucleotide exchange. Humans and mice have three Galphai isoforms although Galphai2 (encoded by Gnai2) and Galphai3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2-/- T and B cells have severe defects in chemokine-receptor signaling. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells fail to properly enter the T cell zone. We have studies the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us to study other hematopoietic cell types. To test the role of Galphai2 and Galphai3 in T cell development we examined Galphai3 deficient mice; mice that deleted Galphai2 in CD4 positive cells; and mice that lacked Galphai3 expression and deleted Galphai2 expression in CD4 positive cells. The loss of Gnai3 had no phenotype, the loss of Gnai2 in CD4 expressing cells led to a mild thymocyte retention phenotype and a reduction in peripheral T cells. Peripheral T cell migration to chemokines was reduced by 40%. The loss of both Galphai2 and Galphai3 in CD4 positive cells led to a severe thymocyte retention phenotype. However, some thymocytes escaped and accumulated within the spleen as mature T cells. These cells exhibited a memory phenotype and most expressed PD-1. Approximately half of the PD-1 positive CD4 T cells expressed CXCR5. Despite their CXCR5 expression they were incapable of migrating to CXCL13 or to any other chemokine. Because of difficulties in generating these mice we performed bone marrow transfers using wild type mice as recipients. These mice had a similar phenotype, but had much more profound peripheral T cell lymphopenia. This suggested that the expansion of PD-1 positive CD4 T cell population that we had observed previously was suppressed by the recipient mouse environment. To test whether radio-resistant T cells in the recipient accounted for the suppressive activity transferred bone marrow into Rag-deficient mice. These mice developed a lymphoproliferative disease with a marked expansion of CD4 positive and PD-1 positive cells that variably express CXCR5. As previously the CD4 T cells in these mice were incapable of migrating to chemokines. As another approach to assessing the role of RGS proteins we have examined mice with a Gnai2 knock-in obtained from Richard Neubig (University of Michigan). This mutation renders Galphai2 resistant to the effect of RGS proteins. These KI mice (G184S) possess a striking phenotype verifying the overall important of RGS proteins in G-protein regulation. They have a marked reduction in peripheral T cells; an increase in mature T cells in the thymus; an absence of many peripheral lymph nodes; disorganized lymphoid organ architecture; splenomegaly; abnormal lymphocyte responses to chemokines; and reduced serum levels of IgG3, but increased levels of Ig2b. Neutrophils from these mice accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background chemoattractant signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Further analysis of B cells from these mice revealed have they markedly elevated basal calcium levels, but poor chemokine induced increases; enhanced non-specific migration, but poor chemotaxis. In striking contrast, the same B cells exhibited enhanced sensitivity to Sphingosine 1-Phosphate (S1P). Analysis of T cells from these mice revealed that RGS/Galphai2 interactions are essential for normal thymocyte egress, T cell trafficking, and homeostasis. Mature KI thymocytes accumulated in the perivascular channel of thymic corticomedullary venules. In the periphery, a severe reduction in nave CD4 T cells and Tregs occurred. The mutant CD4+ T cells adhered poorly to high endothelial venules and exhibited defects in lymph node entrance and egress. The kinetics of chemokine receptor signaling were disturbed, including chemokine-induced integrin activation. Despite the thymic and lymph node egress defects, sphingosine-1-phosphate signaling was not obviously perturbed. This study identified a RGS protein dependent step needed for thymocyte reverse transmigration, and illuminated the importance of RGS proteins in T cell trafficking and function. RGS proteins work in conjunction with the beta-arrestin proteins to regulate chemokine and sphingosine-1 phosphate receptor signaling in lymphocytes. To assess the importance of beta-arrestin1 and beta-arrestin2 in lymphocyte function we have acquired mice that lack either beta-arrestin1 or beta-arrestin2. The analysis of these mice suggest significant problems in the sphingosine-1 phosphate receptor 1 (S1PR1) signaling pathway. Intriguingly the humoral immune response is significantly impaired in the beta-arrestin2 mice. To explore the interaction between the S1PR1 receptor and the beta-arrestin proteins we have established a BRET assay, which will be used to assess the recruitment of these proteins to S1PR1 and to the chemokine receptor CXCR5, one of the major B cell chemoattractant receptors. Besides Galphai proteins B lymphocytes also strongly express Galphas. To assess the importance of Galphas signaling in B lymphocytes we have acquired mice that have a floxed Galphas allele. These mice have been backcrossed on to a C57Bl/6 background and then crossed to mice with a knock-in of the Cre recombinase under the control of the mb1 regulatory sequences. This allows for a B lymphocyte specific deletion of Galphas. These mice are now available for analysis. Preliminary results suggests problems in B cell development and differentiation in the spleen. Further functional studies are in progress. Finally, we examined the role of Galphai proteins in the follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen. Mice lacking B cell expression of Galphai2 and Galphai3 exhibit a striking decrease in MZ B cell development. We found that direct contact with Notch ligand expressing stromal cells (OP9-DL1) could not rescue this developmental defect. Briefly treating wild type B cell progenitors with pertussis toxin, which irreversibly blocks subsequent Galphai nucleotide exchange, inhibited the appearance of MZ B cells in OP9-DL1 cultures. Revealing faulty Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) dependent Notch2 processing, Galphai deficient transitional B cells constitutively had low membrane ADAM10 expression and reduced levels of Notch2 target genes despite normal Notch2 levels. Immunoblotting B cell lysates from Galphai deficient B cells documented a defect in the processing of ADAM10 to its mature form. Suggesting that Galphai signaling promotes ADAM10 membrane expression, stimulating wild type transitional B cells with CXCL12 raised it. These results showed that Galphai signaling supports ADAM10 maturation and activity in developing splenic B cells, and ultimately Notch2 signaling to help drive MZ B cell development.